Generation of the 3-D Caco-2 Large Intestinal Cell Model
The colorectal adenocarcinoma (Caco-2) cell line was obtained from the American Type Culture Collection (ATCC HTB-37™, ATCC, Manassas, VA). Cell monolayers were propagated in 75 cm2 tissue culture flasks at 37°C and 5% CO2 until they were approximately 90% confluent. Tissue culture media consisted of MEM alpha (containing L-glutamine, vitamins, ribonucleosides, and deoxyribonucleosides, but not sodium bicarbonate; Life Technologies, Carlsbad, CA), 2.2 g/L NaHCO3 (Sigma Chemical Company, St. Louis, MO), 20% heat inactivated fetal bovine serum (Sigma), 0.5 μg/mL Fungizone® (Invitrogen, Carlsbad, CA), and 100 U each of penicillin and streptomycin (Invitrogen). Cells were trypsinized and resuspended in 10 mL MEM alpha with 20% heat inactivated FBS and enumerated using a Coulter Counter (Beckman Coulter, Fullerton, CA). A minimum of 2 × 106 cells (total cell count) was used to seed the bioreactor.
Approximately 250 mg of Cytodex-3 porous collagen-I coated microcarrier beads (Sigma) were washed 3X in 1X Hanks Balanced Salt Solution without calcium and magnesium (HBSS, Sigma). On the final wash the HBSS was aspirated to approximately 1 mL above the bead suspension, and then transferred to a 55 mL reusable slow turning lateral vessel bioreactor (Synthecon, Houston, TX) fitted with 5 and 10 mL leur-lock type syringes. The hydrated bead suspension volume was approximately 2.5 mL. Cells were then transferred to the bioreactor and incubated in a 37°C, 5% CO2
incubator without rotation for 1 hour to allow cells to attach to the microcarrier beads. Media was then added to the bioreactor through the 5 and 10 mL syringes until the bioreactor was full, and the 5 mL syringe was nearly filled with media. Air bubbles in the bioreactor were expelled through the 5 and 10 mL syringes. When free of bubbles, the stopcocks between the syringes and bioreactors were closed and the bioreactor was attached to the base and the rotation speed was set at 18 RPM. The bioreactor was inspected daily for air bubbles, and if detected, the bioreactor was detached from the base, the stopcocks opened, and bubbles expelled. After inspection, the bioreactor was attached to the base and returned to its normal rotational speed. Approximately 90% of the media was exchanged after 5 days, and then exchanged daily for 13-16 more days. After 18-21 days in the bioreactor, the mature aggregates were transferred to a 50 mL conical centrifuge tube and washed 3 times with 1X HBSS. Aggregates were then transferred to 24-well tissue culture plates (50 μL per well, approximately 1 × 105
cells/mL) using a micropipettor fitted with a wide bore pipette tip. Some aggregates were transferred to 1 mL microcentrifuge tubes and characterized for markers of cellular differentiation as previously described (Honer zu Bentrup et al. 2006
; Nickerson et al. 2001
; Straub et al. 2007
). Trypan Blue dye exclusion was performed to assess the viability of the cells prior to infection. Greater than 95% of the cells were viable after culture for 18-21 days. In addition, some aggregates were further characterized by examination of ultra-thin sections using transmission electron microscopy. Depending on the cell yield, three to four 24-well plates were produced from one bioreactor.
Human norovirus stocks were obtained from several sources. Genogroup I.1 (GenBank Accession Number M87661) viruses were kindly provided by C.L. Moe at Emory University from a previous human challenge trial. Stool samples from two anonymous patients (denoted as Patient 3 and Patient 4), collected pre and post challenge with GI.1, were identified as hNoV negative (3-1, and 4-1) and positive stool samples (3-9 and 4-3), respectively. Genogroup II samples, identified as sample 386 and 1G, were kindly provided by C.P. Gerba at the University of Arizona and were obtained from various outbreak settings. Sequencing data of the RNA dependent RNA polymerase gene, provided by the University of Arizona, indicated that strain 386 is most closely related to GenBank EF126966 (Norovirus Hu/GII.4/Nijmegen115/2006/NL) and 1G is most closely related to GenBank AB240184 (Norovirus Hu/NV/Hokkaido/284/2004/JP).
All hNoV samples were resuspended in 1X PBS as 10% (w/v) slurry. The samples were vigorously vortexed, and then the solids were pelleted by centrifugation at 3,000 x g for 10 min. The supernatant was filtered through a 0.2 μm pore size filter. From the filtrate, 50 μL of virus was processed for quantitative reverse transcription real-time PCR (herein abbreviated as qRT-PCR) using a Qiagen RNEasy Plus kit (Qiagen, Valencia, CA) per manufacturer’s instructions. Details for viral enumeration by qRT-PCR follow.
For infectivity assays a 1:1,000 dilution of the viral stocks (and hNoV negative stool samples), prepared in sera free MEM alpha, was used to infect the aggregates in the 24-well tissue culture plates. The predicted initial quantity of virions in these dilutions, determined by qRT-PCR, were as follows. For the two different genogroup I samples, the initial predicted titer added to each well was 802 ± 58 and 6,390 ± 681 copies for samples 3-9 and 4-3, respectively. For the Genogroup II viruses, 386 had an initial predicted titer of 41 ± 7 copies and 1G had a predicted titer of 529 ± 59 copies. The total volume in each well was approximately 150 μL (100 μL of sample and 50 μL of cells). This volume was sufficient to just cover the cells and allow for maximal viral contact with the cell suspension. Plates were incubated for 1 hr at 37°C in a 5% CO2 incubator. Plates were gently rocked every 15 minutes.
For the 1 hr and all subsequent time points, wells were completely sampled, without replacement, at each time point. For any given time point, some wells would be processed to assess viral RNA amplification and others for microscopic analysis. At 1 hr post infection, cells in all wells were overlaid with 1 mL of sterile sera free MEM alpha. After overlaying the cells with media, wells designated as the 1 hr post infection time point were harvested using a micropipettor fitted with a wide bore pipette tip to maintain tissue integrity, and transferred to 1.5 mL microcentrifuge tubes. Cells were centrifuged at 1,500 x g for 3 min and the supernatant was aspirated and discarded. A portion of the tissue aggregates was flash frozen in liquid nitrogen and stored at -80°C for qRT-PCR. The qRT-PCR copies at 1 hr post infection in the tissue aggregates was the observed titer that had attached to the cells and this was used as a baseline when calculating viral RNA amplification. Another portion of the tissue aggregates was fixed with 2.5% glutaraldehyde in 1X PBS and stored at 4°C for further analysis by transmission electron microscopy. This same harvesting process was repeated for subsequent time points up to 1-week post infection.
Microscopic Analysis of Infection
Initial observation of the uninfected and infected tissue aggregates for cytopathic effect (CPE) was made using a Micromaster® inverted microscope with phase contrast infinity corrected optics (Westover Scientific, Mill Creek, WA). Images were captured and saved as jpeg images using a Micron USB2 Camera and Software (Westover Scientific). The tissue aggregates were examined for potential cytopathic effect (CPE) before harvesting the cells for further examination by TEM or qRT-PCR.
Transmission Electron Microscopy of Tissue Thin Sections
Sub-cellular CPE was assessed by transmission electron microscopy using the methods described by Straub et al
. (Straub et al. 2007
). Ultra-thin sections (70 nm) were affixed on 100 mesh copper grids coated with forvar and carbon (Electron Microscopy Sciences (EMS), Hatfield, PA) and were post-stained with 1% aqueous uranyl acetate (EMS) and 1% Reynolds lead citrate (Reynolds 1963
) before viewing on a Tecnai T-12 transmission electron microscope (FEI Co., Hillsboro, OR) operating at 120 kV with LaB filament. Images were collected digitally using a 2x2K UltraScan 1000 charge-coupled device (Gatan Inc., Pleasanton, CA).
Immune Electron Microscopy
Immune electron microscopy (IEM) was performed on GI.1 infected (4-3) and uninfected tissues (4-1). For these experiments, IEM was performed on viruses that had been passaged through the 3-D Caco-2 cell cultures two times. After freeze thaw lysis and extraction with decafluropentane, the titer measured by qRT-PCR for the infected tissues after passage was 1.01×106 copies/mL. The primary antibody was a mouse monoclonal antibody to a peptide in the GI.1 capsid (P2B2; Abcam; Cambridge, MA), and the secondary antibody consisted of 6 nm gold particles conjugated with goat anti-mouse IgG (EMS). Controls for this procedure consisted of 1) uninfected cells and 2) infected cells with the primary antibody omitted.
To ensure that viruses were cell-associated, harvested tissue aggregates were washed 3X in sterile 1X PBS. Briefly, tissue aggregates, free of media, were resuspended in PBS, gently pelleted by centrifugation (1,500 x g for 3 min.), and the supernatant aspirated. This process was repeated two additional times. After the 3rd wash, the supernatant was aspirated and the pellets were resuspended in 500 μL of 1X PBS. Viruses were released from the cells by freezing at -80°C and thawing at room temperature. The crude lysate was extracted with an equal volume of Vertrel (decafluropentane; Sigma, St. Louis, MO), and the aqueous phase was recovered. Uninfected cells were processed in the same manner. The aqueous phase was then processed for IEM.
Processing of the EM grids consisted of a series of different buffer and antibody solutions arrayed on a hydrophobic piece of Parafilm (VWR), and all steps were performed at room temperature. After the TEM grids were exposed face down to drops of hNoV extracts for 10 min, they were processed by transfer to 100 μL drops of respective solutions by the following protocol: fixation in 2% paraformaldehyde in 1X PBS (EMS) for 15 min, 3 washes in 1X PBS, (2 min each), 1% bovine serum albumin (BSA) in 1X PBS (3 min), blocking in 2% BSA (20 min), and a wash with 1% BSA (3 min). For grids receiving primary antibody, the optimal dilution was determined to be 1:100 in 1% BSA. For controls where primary antibody was omitted, grids were exposed to 1% BSA only. Primary antibody exposure (or mock) proceeded for 60 min. The grids were then washed 3 times in 1% BSA, exposed to secondary antibody, (1:20 in 1% BSA, 60 min), washed 3x in 1% BSA and then 3x in 1X PBS. The grids were post-fixed in 2% glutaraldehyde in 1X PBS (EMS) for 5 min, finally washed 3x in 1X PBS and 3x in distilled water. Grids were counterstained with Nano-W (Methylamine Tungstate, Nanoprobes, Yaphank, NY) for 30 s, blotted dry, and then imaged.
Nucleic Acid Extraction From Stools and Cells
To prepare stool samples and tissue aggregates for qRT-PCR analysis, materials were processed using Qiagen’s RNEasy Plus kit (Qiagen, Valencia, CA). The protocol was modified to include the use of a QiaShredder column (Qiagen) and a genomic DNA removal column allowing total RNA to be analyzed for virus or host RNA. Tissue aggregates (50 μL with some residual liquid) were suspended in 350 μL of buffer RLT containing 0.146% beta mercaptoethanol (ß ME, 10 μL of a 14.6% solution added to each mL of buffer RLT) and vortexed until the microcarrier beads dissolved. The suspension was homogenized by passing it through a QiaShredder column (Qiagen). The filtrate was then added to the genomic DNA removal column. The filtrate was recovered, and the remainder of the procedure per manufacturer’s instructions, was followed exercising the following options. After the last wash with buffer RPE, the columns were transferred to clean collection tubes and centrifuged at 16,100 x g for 1 min to ensure ethanol removal. The column was eluted with nuclease-free distilled water (2 × 30 μL) yielding a final volume of approximately 60 μL (approximately the same volume starting the procedure). This allowed direct comparison of viral copy number in either stool or cellular pellets without mathematical conversion (e.g. raw copy number at 1 hr post infection could be directly compared to copy number at 1 week post infection). RNA concentration was determined using a NanoDrop 1000 spectrophotometer (Thermo Fisher Scientific, Wilmington, DE). Human GAPDH and STAT-1 were also measured by qRT-PCR as a secondary assessment of host RNA concentration for other studies. For human GAPDH and STAT-1, validated qPCR kits were purchased from Applied Biosystems (Applied Biosystems, Foster City, CA), and after cDNA synthesis, qPCR was performed on the ABI 7500 Fast Real-time PCR System (Applied Biosystems). For uninfected cells, total RNA recovery was roughly consistent regardless of time point. For infected cells, total RNA concentration increased as a function of increased viral RNA copies in the cells over time ().
Measured RNA concentrations in uninfected and infected cells.
Quantitative PCR Standards
Amplification of the PCR products to produce qRT-PCR standards, cloning the amplicons into plasmids, generation of RNA transcripts, and purification of the transcripts employed standard protocols provided by kits from various suppliers. The Genogroup I standard transcript is based on sample 155 (GI.1) from Straub et al
. (Straub et al. 2007
), while the Genogroup II was based on sample VS122 (provided by C.P Gerba from a previous study). PCR primers () were designed to amplify a fragment that was slightly longer than the segment amplified by real-time PCR. Degenerate primers were used to generate the standards due to the high amount of sequence diversity in hNoV strains.
PCR primers to generate real-time PCR standards for human norovirus genogroups GI and GII.
Quantitative Reverse Transcription PCR
Quantitative RT-PCR for Genogroup I and Genogroup II followed the protocol of Kageyama et al
. (Kageyama et al. 2003
), with the following modifications. Real-time PCR was performed on an ABI 7500 Fast Real-Time PCR System, but using a modified thermal cycling protocol (2 min at 50°C for Uracil DNA Glycosylase digestion, 10 min at 95°C, and then 45 cycles of denaturation for 15 sec at 95°C and primer annealing and extension for 1 min at 56°C), and standards for Genogroup I and Genogroup II were generated from in vitro
RNA transcripts as described above.
For standards, mock infected, negative stool infected, hNoV infected, and no template controls, reverse transcription was performed using SuperScript III reverse transcriptase following the manufacturer’s recommended protocol (Invitrogen, Carlsbad, CA). From the RT reaction, 2 μL of cDNA (or 2 μL of nuclease free water for no template controls) was used in each real-time PCR reaction. Primer and probe concentrations and thermal cycling protocols were identical to Kageyama et al
. (Kageyama et al. 2003
), with the exceptions noted above. The Kageyama protocol was ultimately chosen because, for environmental sampling, flexibility is required to detect any potential GI and/or GII strains. Their protocol targets the most conserved region in all known norovirus genomes (e.g the ORF1 ORF2 junction). To quantitatively measure and compare viral RNA amplification within an infection trial, all time points were run on the same 96-well plate as the standards and no-template controls. This was because the Kageyama protocol typically had an efficiency of 85 to 100% (slope = -3.7 to -3.3 for 85 and 100% efficiency, respectively). By running an entire experiment on one plate, the variability in PCR amplification efficiency could be mitigated. Thus, even if the amplification efficiency was low, log increases in viral amplification could still be assessed because they were compared against the same standard curve. In addition, PCR inhibition from the samples could also be assessed by comparing the slopes of the samples with the slope of the slopes of the standards (Applied Biosystems Technical Communication). During the exponential phase of amplification, equivalent slopes of the samples and standards indicated that there is no PCR inhibition in the processed samples (). All time points, standards and no template controls were performed in triplicate.
Figure 1 Logarithmic amplification plot of a quantitative reverse transcription real-time PCR experiment comparing hNoV infected Caco-2 cells with standards. For clarity only the 107 and 106 copy standards are displayed. The data for the infected cells is presented (more ...)